Hemoglobin present in red blood cells can be glycated by the non-enzymatic addition of a glucose molecule to the amino terminus of the β-chain of the hemoglobin. Once a hemoglobin molecule is glycated, it remains glycated, and an accumulation of glycated hemoglobin within a red cell reflects the average level of glucose to which the cell has been exposed during its life cycle. The level of glycated hemoglobin present in an individual's blood is thus proportional to the level of glucose in the blood and is an indicator of the individual's mean daily blood glucose concentration over the previous four weeks to three months. The ratio of glycated hemoglobin to total hemoglobin in a whole blood sample is therefore quite useful in the diagnosis and monitoring of patients with diabetes mellitus.
Numerous methods exist for determining the level of glycated hemoglobin in human blood, most of which involve calculating the relative amount of glycated hemoglobin A (HbA1c) present in the blood, as hemoglobin A (HbA) is the major form of hemoglobin present in human blood. There are several methods currently used to detect HbA1c and Hb to determine glycemic control. Techniques such as high performance liquid chromatography and immunoaffinity selection are used in such methods, which take advantage of physical and/or chemical properties of glycated hemoglobin A that distinguish it from other forms of hemoglobin present in the blood. In the HPLC method, a whole blood sample is centrifuged, the cellular fraction lysed, and the hemoglobin fractions separated using an anion exchange HPLC column and an absorbance measurement. Other chromatographic methods like phenyl-boronate chromatography separation, where only HbA1c binds to the column and non-glycated Hb comes through, have also been used. Two other cartridge based methods use a combination of chemistries and/or immunoassays to measure Hb and HbA1c (e.g., DCA VANTAGE®, Siemens Healthcare Diagnostics Inc., Tarrytown, N.Y.; and AFINION™ HbA1c, Axis-Shield PoC AS, Oslo, Norway). In these cases, whole blood is lysed, and samples are reacted with a combination of dry and liquid reagents which are read spectrophotometrically.
However, the currently used methods require separation of the whole blood sample and detection of HbA1c and total hemoglobin in separate steps. Therefore, a need exists for new and improved assay methods for determining the ratio of glycated hemoglobin to total hemoglobin in a whole blood sample, as well as kits and microfluidics devices useful in said methods. It is to such assays that minimize sample processing, reduce the number of assay steps and reagents, and use a single technology to detect both HbA1c and Hb, as well as kits, devices, and methods related thereto, that the presently disclosed and/or claimed inventive concept(s) is directed.